Endothelial cell (EC) injury has been widely documented in certain infections, autoimmunity, allograft rejection, graft versus host disease and following immunotherapy with cytokines or immunotoxins. It is becoming increasingly clear that ECs are primary targets of immunologic attack, although the precise mechanism remains unclear. We have used interleukin-2 (IL-2)-induced vascular leak syndrome (VLS) as a model to study EC injury. IL-2 is the only FDA approved systemic therapy for treating metastatic renal cell carcinoma. It is also being used to treat metastatic melanomas, immunodeficiencies and viral infections including AIDS. Despite much success, the efficacy is limited by severe, life-threatening toxicity resulting from EC injury leading to VLS. Using CD44 knockout (KO), perforin KO and FasL mutant mice, we have demonstrated that IL-2 activates cytotoxic lymphocytes and such cells use CD44 to kill EC by triggering perforin and FasL. More recently, we have demonstrated that CD44v7 exon KO mice also exhibit decreased VLS and EC killing. Based on these studies, we will test the hypothesis that IL-2 treatment up regulates CD44 variant isoforms on cytolytic lymphocytes of which v7 isoform plays a key role in EC injury and induction of VLS. The specific aims are 1) To test the susceptibility of EC isolated from various organs to cytotoxicity mediated by IL-2 activated T/NK/NKT cells from WT, pefforin KO, Fas and FasL mutant mice. Using NK cell-deficient (NKCD-Tg) mice, the role of NK cells in IL-2 induced VLS will also be investigated. 2) To examine whether down regulation of killer cell inhibitory receptor (KIR)-like molecules and/or up regulation of stimulatory receptors on NK cells is responsible for CD44-mediated EC injury. Specifically, the expression of KiR-like molecules, including Ly49 and CD94/NKG2 present on NK cells, and the classical and non-classical MHC ligands on Ecs, will be studied. 3) Identification of the CD44 isoforms involved in EC injury caused by cytotoxic lymphocytes. 4) To address the mechanism by which CD44v7KO mice exhibit increased resistance to VLS. 5) Use of mimetics of CD44 or its ligand including mAbs against CD44s and CD44v isoforms, Pep-l(an inhibitor of HA), CD44Rg and CD44MutRg fusion proteins to prevent EC injury and vascular leak. Together, the current study should provide novel insights into the mechanism of immune cell-mediated EC injury and development of strategies to prevent vascular leak.